Project description:Leafy spurge is a model for studying well-defined phases of dormancy in underground adventitious buds (UABs) of herbaceous perennial weeds, which is a primary factor allowing many invasive perennial weeds to escape conventional control measures. A 12-week ramp down in both temperature (27M-BM-0C M-bM-^FM-^R 10M-BM-0C) and photoperiod (16 h M-bM-^FM-^R 8 h light) is required to induce a transition from para- to endo-dormancy in UABs of leafy spurge. To evaluate the effects of photoperiod and temperature on molecular networks associated with this transition, we compared global transcriptome data-sets obtained from UABs of leafy spurge exposed to a ramp down in both temperature and photoperiod (RDtp) vs. a ramp down in temperature (RDt) alone. Analysis of transcriptome data-sets indicated that numerous genes associated with circadian clock, photoperiodism, flowering, and hormone responses (CCA1, COP1, HY5, MAF3, MAX2) were preferentially expressed during the transition from para- to endo-dormancy. Gene-set enrichment analyses highlighted metabolic pathways associated with ethylene, auxin, flavonoids, and carbohydrate metabolism; whereas, sub-network enrichment analyses identified hubs (CCA1, CO, FRI, mir172A, EINs, DREBs) of gene networks associated with carbohydrate metabolism, circadian clock, flowering, and stress and hormone responses during the transition to endodormancy. These results helped refine existing models for the transition to endodormancy in UABs of leafy spurge, which strengthened the roles of circadian clock associated genes, DREBs, COP1-HY5, carbohydrate metabolism, and involvement of hormones (ABA, ethylene, and strigolactones). Further, we propose that the RDtp treatment ultimately leads to a chain effect, responsive to photoperiod and temperature signaling, to synchronize molecular processes associated with the transition from para- to endo-dormancy. Plant material, environmental treatments, and vegetative growth Leafy spurge plants were propagated from the uniform biotype (1984-ND001) and maintained in a greenhouse as described by Anderson and Davis (2004). Prior to the start of each experiment, plants were acclimated in a Conviron growth chamber (Model PGR15) for one week at 27M-BM-0C, 16:8 h light:dark photoperiod. Each experiment was replicated four times, and each replicate contained 30 plants. Six plants from each replicate were used to determine vegetative growth rate, and crown buds from the remaining 24 plants were collected for transcriptome studies. All samples were collected between 11:00 and 13:00 a.m. to avoid diurnal variation. We previously established conditions for induction and release of dormancy phases under controlled environments (DoM-DM-^_ramacM-DM-1 et al. 2010; Foley et al. 2009). To induce endodormancy, paradormant plants were subjected to a ramp-down in temperature (27M-BM-0C M-bM-^FM-^R 10M-BM-0C) and photoperiod (16 h M-bM-^FM-^R 8 h light), i.e., RDtp, for 12 weeks (Fig. 1, Exp-1). To distinguish the individual effects of temperature and photoperiod on molecular networks involved in endodormancy induction (see Fig. 1, Exp-2), we compared three-month old paradormant plants subjected to a ramp-down in temperature (27M-BM-0C M-bM-^FM-^R 10M-BM-0C) under constant photoperiod (16 h light) for 12 weeks (i.e., RDt) to RDtp plants. Additionally, a set of paradormant plants were kept under constant temperature and light (27M-BM-0C, 16 h light) as a control. At the end of each treatment, the aerial portion of the plant was decapitated to determine the dormancy status of the crown buds by their vegetative growth rate in the greenhouse, as described by Foley et al. (2009). New vegetative shoot growth was recorded weekly; results were analyzed using the generalized linear mixed model (PROC GLIMMIX) procedure of SAS 9.2, and 95% confidence intervals for treatment by week means were generated. Transcriptome analyses At the end of each treatment, crown buds were collected from intact plants to study transcriptome profiles and were stored at -80M-BM-0C. RNA extraction, cDNA synthesis and fluorescent labeling, microarray hybridization using ~23K element arrays, and spot intensity analyses were performed as previously described (DoM-DM-^_ramacM-DM-1 et al. 2010). Transcriptome data obtained from Exp-1 and -2 (Fig. 1) was used for various bioinformatics analysis. GeneMaths XT 2.1 software was used to normalize the arrays, to conduct statistical analyses, and to generate Venn diagrams as previously described by DoM-DM-^_ramacM-DM-1 et al. (2010). Expression data for both Exp-1 and -2 are deposited at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) as GEO dataset query GSE19217 and GSE??, respectively. The entire data set was further analyzed using Ariadne Pathway Studio 7 Software-Resnet Plant Version 2.1 (Ariadne Genomics Inc., Rockville, MD, USA) to obtain Gene Set Enrichment Analysis (GSEA) and Sub Network Enrichment Analysis (SNEA). GSEA is used to determine if predefined sets of genes are over-represented (P<0.05) between treatments; focusing on gene sets based on AraCyc metabolic pathways http://pmn.plantcyc.org/ARA/class-instances?object=Pathways, and gene ontology (GO) (http://www.geneontology.org/). SNEA algorithm was used to identify the networks by highlighting over-represented ontologies based on published gene regulation hierarchies, protein:protein interactions, or protein modification targets. Furthermore, these networks were visualized using the Union Selected Pathway function of the software for expression targets, binding partners, protein modification targets, and also custom advanced function to generate sub-networks as neighbors of proteins based on expression, regulation, molecular transport, protein modification, promoter binding, molecular synthesis, chemical reaction, and direct regulation.

Project description:Leafy spurge (Euphorbia esula) is an herbaceous perennial weed that produces vegetatively from an abundance of underground adventitious buds. The objectives of this study were to determine how mimicking natural seasonal conditions (photoperiod and temperature) under controlled environmental conditions affect dormancy and flowering competence; to determine molecular mechanisms associated with well-defined phases of seasonal dormancy transitions based on transcript profiles obtained by microarray analysis; and to link mechanisms regulating induction and release of endodormancy and flowering competence. Reduction in temperature (27 to 10Â°C) and photoperiod (16 to 8 h) over a three-month period induced a para- to endo-dormant transition in crown buds. An additional eleven weeks of prolonged cold (5-7Â°C) and short-photoperiod treatment resulted in accelerated shoot growth from crown buds, and 99% floral competence when plants were returned to growth promoting conditions. Exposure of paradormant plants to short-photoperiod and prolonged cold treatment alone had minimal affect growth potential or on flowering (~1%); whereas endodormant crown buds without prolonged cold treatment, had delayed shoot growth and approximately 2% flowering when returned to growth promoting conditions. Transcriptome analyses revealed that 373 and 260 genes were differentially expressed (p<0.005) during para- to endo-dormant and endo- to eco-dormant transitions, respectively. Transcripts from flower competent vs. non-flower competent crown buds identified 607 differentially expressed genes, and genes involved in cell cycle and DNA processing, oxidative stress, flower regulation, and proteolysis were over-represented. Further, sub-network analysis identified expression targets and binding partners associated with circadian clock, dehydration/cold signaling, phosphorylation cascades, and response to abscisic acid, ethylene, gibberellic acid, and jasmonic acid, suggesting these central regulators affect well-defined phases of dormancy. Potential genetic pathways associated with these dormancy transitions and flowering were used to develop a proposed conceptual model. Leafy spurge is an herbaceous perennial weed that undergoes vegetative reproduction from an abundance of underground adventitious buds which exhibit phases of para-, endo-, and eco-dormancy (defined by Lang et al. 1987) during summer, fall, and winter, respectively (Anderson et al. 2005). In this study a population of leafy spurge plants were propagated through random cuttings from the genetically uniform biotype 1984-ND001. Paradormant crown buds from three-month old greenhouse plants were collected after one week of acclimation under growth chamber conditions. Induction of endodormancy in crown buds was accomplished by subjecting greenhouse grown and growth chamber acclimated plants to a ramp down (RD) treatment consisting of a reduction in temperature of 1.42Â°C week-1 (27Â°C â 10Â°C) and a decreasing photoperiod of 40 min week-1 (16h â 8h light) for 12 weeks as previously established by Foley et al. (2009). These conditions mimic the average seasonal environmental conditions experienced in Fargo, ND (46Â°54â² N, 96Â°48â² W) during the transition from para- to endo-dormancy (Anderson et al. 2005). To induce a transition of crown buds from endo- to eco-dormancy, plants subjected to the RD treatment were given prolonged cold treatment for 11 weeks at 5-7Â°C, under constant 8 h:16 h day:night cycle; light fluencies were approximately 250 Î¼mol m-2 s-1. To compare the effects of the prolonged cold with or without RD treatment on dormancy status and flowering competence in crown buds, greenhouse-grown and growth chamber acclimated plants were subjected directly to an extended cold treatment for 11 weeks at 5-7Â°C, as previously described, without a RD treatment. For microarray hybridizations, labeled cDNAs were prepared from 30 Âµg of total RNA using the Alexa Fluor cDNA labeling kit (Invitrogen, Carlsbad, CA, USA) according to manufacturer's protocols. Labeled cDNAs were hybridized to a custom made 23K element microarray that contained 19,808 unigenes from a leafy spurge EST database (Anderson et al. 2007) and an additional 4,129 unigenes from a cassava EST database (Lokko et al. 2007). Comparison of gene expression between samples was accomplished using a rolling circle dye swap hybridization scheme (Churchill 2002) to provide every biological replicate with technical replicates. In our microarray analyses experiment, each of the four biological replicates included four technical replicates using two different dyes, resulting in a total of 16 technical replicates for each treatment. Microarray hybridization was visualized using a GenePix 4000B scanner and probe intensities and background were quantified using GenePix 6.0 software (Molecular Devices, Sunnyvale, California, USA). A quality control value of â1â was assigned to all probes that had intensity values greater than 2 times the standard deviation over average of the negative control and empty probe intensities (after deletion of 1% of the most intense negative/empty probe values). Hybridization intensities were log2 transformed, and arrays were centered and normalized against each other.

Project description:Leafy spurge (Euphorbia esula) is an herbaceous perennial weed that produces vegetatively from an abundance of underground adventitious buds. The objectives of this study were to determine how mimicking natural seasonal conditions (photoperiod and temperature) under controlled environmental conditions affect dormancy and flowering competence; to determine molecular mechanisms associated with well-defined phases of seasonal dormancy transitions based on transcript profiles obtained by microarray analysis; and to link mechanisms regulating induction and release of endodormancy and flowering competence. Reduction in temperature (27 to 10°C) and photoperiod (16 to 8 h) over a three-month period induced a para- to endo-dormant transition in crown buds. An additional eleven weeks of prolonged cold (5-7°C) and short-photoperiod treatment resulted in accelerated shoot growth from crown buds, and 99% floral competence when plants were returned to growth promoting conditions. Exposure of paradormant plants to short-photoperiod and prolonged cold treatment alone had minimal affect growth potential or on flowering (~1%); whereas endodormant crown buds without prolonged cold treatment, had delayed shoot growth and approximately 2% flowering when returned to growth promoting conditions. Transcriptome analyses revealed that 373 and 260 genes were differentially expressed (p<0.005) during para- to endo-dormant and endo- to eco-dormant transitions, respectively. Transcripts from flower competent vs. non-flower competent crown buds identified 607 differentially expressed genes, and genes involved in cell cycle and DNA processing, oxidative stress, flower regulation, and proteolysis were over-represented. Further, sub-network analysis identified expression targets and binding partners associated with circadian clock, dehydration/cold signaling, phosphorylation cascades, and response to abscisic acid, ethylene, gibberellic acid, and jasmonic acid, suggesting these central regulators affect well-defined phases of dormancy. Potential genetic pathways associated with these dormancy transitions and flowering were used to develop a proposed conceptual model.

Project description:Persistence of the invasive perennial weed leafy spurge is mainly attributed to seasonal production of underground adventitious buds (UABs), which undergo well-defined phases of dormancy (para-, endo- and eco-dormancy). These well-defined phases of dormancy also allow UABs of leafy spurge to survive extreme seasonal variations, including dehydration-stress. Consequently, objectives of this study include understanding the effects of dehydration-stress on vegetative reproduction, flowering competence, and transcript profiles at different phases of bud dormancy. The vegetative growth potential of UABs was monitored by removing the aerial portion of dehydration-stressed plants and re-watering the root system. Further, microarray analysis was used to follow transcriptome profiles to identify critical defense- and signaling-pathways at different phases of dormancy in UABs. Surprisingly, only 3 days of dehydration-stress is required to break the endodormant phase in UABs. In leafy spurge, vernalization of endodormant UABs has previously been shown to induce flower competence, while breaking of endodormancy via dehydration-stress did not induce floral induction. Thus, these two environmental treatments open a unique approach to independently dissect molecular mechanisms involved in endodormancy maintenance and floral competence. Bioinformatics analysis of transcriptome data helped to identify models and overlapping pathways. During endodormancy break, LEC1, PHOTOSYSTEM I RC, and brassinosteroids were identified as ooverlapping hubs of up-regulated genes, and DREB1A, CBF2, GPA1, MYC2, BHLH, BZIP, and flavonoids were identified as overlapping hubs of down-regulated genes. Additionally, key genes involved in metabolic activity, chromatin modification, and cross-talk between growth regulators were identified as playing a role during endodormancy maintenance. Plant material, controlled environmental treatments, and vegetative growth: Three-month-old paradormant leafy spurge plants grown in a greenhouse were acclimated for one week, under growth chamber conditions, prior to being subjected to a ramp-down in temperature (27 °C → 10 °C) and photoperiod (16 h → 8 h light) for 12 weeks to induce endodormancy, as previously established (Doğramacı et al. 2010; Foley et al. 2009). Endodormancy status was confirmed by comparing vegetative growth rates of plants with and without a ramp-down treatment. To study the effects of dehydration-stress on vegetative growth from UABs, water was withheld from endodormant plants up to 21 days. After dehydration for 0, 1-, 3-, 7-, 14-, and 21-days, the aerial portion of the plants were decapitated at the soil surface and vegetative growth and floral competence of UABs were monitored under growth-conducive conditions by re-watering the root system. Vegetative shoot growth from six plants was recorded weekly for four weeks, and results of four replications were analyzed using SAS 9.2 (SAS Institute, Cary, NC, 2008) software as described by Doğramacı et al. (2010). Microarray analysis: At the end of each treatment, crown buds were collected, and RNA extraction, cDNA synthesis, fluorescent labeling, microarray hybridization using ~23K element arrays, and spot intensity analyses were performed as previously described by Doğramacı et al. (2010). Based on unexpected vegetative growth results obtained from this study, only microarray data obtained from 0-, 1-, and 3-day dehydration-stressed endodormant UABs, which were compared with microarray data for endodormant, and flowering competent ecodormant UABs from our previous study (Doğramacı et al. 2010; GSE19217), were used for bioinformatics analyses. However, to merge the datasets from these two separate experiments, T-tests were first performed on microarray data obtained from endodormant UABs from each experiment, and differentially-expressed genes (p<0.005) were removed from the dataset (~5% of the spots). After removing the outliers, the two endodormant transcriptome data sets were grouped together and used as the baseline control for further ANOVA, T-tests and other analyses. Array normalization, statistical analyses and clustering of the dataset and Venn diagram generation were done using GeneMaths XT 5.1 software as previously described by Doğramacı et al. (2010).

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Manipulation of Temperature and Photoperiod Identifies Molecular Networks Associated with the Para- to Endo-dormant Transition in Leafy Spurge Crown Buds

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